The nature of biochemically synthetic bilirubin sulfate was examined on detail, and bilirubin sulfate and bilirubin glucuronide were measured in bile of heterozygote Gunn rat and Wister strain rat. Further, after the intrvenous injection of (3)H-bilirubin sulfate into heterozygote Gunn rats and Wister strain rats, the biliary excretion rate of bilirubin sulfate was studied in them. The following results were gained by these studies. 1) The absorption peak was 452 nm in the synthetic bilirubin sulfate solution, pH 2.2, and its azo pigment showed each peak absorptions at 535-545 nm in pH 2.0 and at 565-575 nm in pH 1.0. The molar ratio between bilirubin and sulfate in the band of thin layer chromatography showing an Rf of 0.26 was determined from 1.72 to 1.96 with a mean of 1.78±0.09 S.D.. These data suggest that this compound is bilirubin disulfate. 2) In the bile of Gunn rats and Wister strain rats, the molar ratio of glucuronic acid to esterfrom bilirubin was inversely proportional to that of sulfate radical to direct bilirubin (r=-0.74, p<0.05). This implies that the conjugation of bilirubin with sulfate increases compensatedly as compared to the disturbance of that of bilirubin with glucuronic acid. 3) The excretion ratios of 2 hours to 24 hours in the intravenous (3)H-bilirubin sulfate loading were 54.2% in Gunn rats and 63.4% in Wister strain rats. This finding supports the concept that bilirubin sulfate is easily excreted in the bile. The biliary excretion within 24 hours following the administration of bilirubin sulfate was 40.4% in Gunn rats and 69.4% in Wister strain rats, and these excretion rates decreased in parallel with the decrease of molar ratio in bilirubin glucuronide (r=0.96, p<0.01). It is suggested that the ability to excrete bilirubin sulfate may be the limiting factor in the existence of defective hepatic bilirubin glucuronidation.